Peroxynitrite-mediated Toxicity Research Articles

Endothelial NO and O2[rad]− production rates differentially regulate oxidative, nitroxidative, and nitrosative stress in the microcirculation

Endothelial dysfunction causes an imbalance in endothelial NO and O2− production rates and increased peroxynitrite formation. Peroxynitrite and its decomposition products cause multiple deleterious effects including tyrosine nitration of proteins, superoxide dismutase (SOD) inactivation, and tissue damage. Studies have shown that peroxynitrite formation during endothelial dysfunction is strongly dependent on the NO and O2− production rates. Previous experimental and modeling studies examining the role of NO and O2− production imbalance on peroxynitrite formation showed different results in biological and synthetic systems. However, there is a lack of quantitative information about the formation and biological relevance of peroxynitrite under oxidative, nitroxidative, and nitrosative stress conditions in the microcirculation. We developed a computational biotransport model to examine the role of endothelial NO and O2− production on the complex biochemical NO and O2− interactions in the microcirculation. We also modeled the effect of variability in SOD expression and activity during oxidative stress. The results showed that peroxynitrite concentration increased with increase in either O2− to NO or NO to O2− production rate ratio (QO2−/QNO or QNO/QO2−, respectively). The peroxynitrite concentrations were similar for both production rate ratios, indicating that peroxynitrite-related nitroxidative and nitrosative stresses may be similar in endothelial dysfunction or inducible NO synthase (iNOS)-induced NO production. The endothelial peroxynitrite concentration increased with increase in both QO2−/QNO and QNO/QO2− ratios at SOD concentrations of 0.1–100μM. The absence of SOD may not mitigate the extent of peroxynitrite-mediated toxicity, as we predicted an insignificant increase in peroxynitrite levels beyond QO2−/QNO and QNO/QO2− ratios of 1. The results support the experimental observations of biological systems and show that peroxynitrite formation increases with increase in either NO or O2− production, and excess NO production from iNOS or from NO donors during oxidative stress conditions does not reduce the extent of peroxynitrite mediated toxicity.

Myricitrin protects against peroxynitrite-mediated DNA damage and cytotoxicity in astrocytes

Peroxynitrite, a potent oxidising and nitrating species, has been implicated in the pathogenesis of neurodegenerative diseases. This study was undertaken to investigate the protective effect of myricitrin on peroxynitrite-mediated toxicity and the underlying mechanism. Our results showed that the presence of myricitrin was found to significantly inhibit peroxynitrite-mediated DNA damage. EPR spectroscopy demonstrated that myricitrin potently diminished the DMPO-hydroxyl radical adduct signal from peroxynitrite. Further study showed that glutathione (GSH) depletion caused by peroxynitrite can be effectively prevented by pre-incubation of astrocytes with myricitrin. Moreover, co-incubation of astrocytes with myricitrin and buthionine sulfoximine (BSO) eliminated the myricitrin-induced GSH increase. In contrast, co-incubation of myricitrin with BSO slightly protected astrocytes against cytotoxicity and DNA damage mediated by peroxynitrite. These results revealed that myricitrin can protect against peroxynitrite-induced DNA damage and cytotoxicity, which might have implications for myricitrin-mediated neuroprotection.

Peroxynitrite Detoxification and Its Biologic Implications

Peroxynitrite is a cytotoxic oxidant formed in vivo from the diffusional-controlled reaction between nitric oxide and superoxide radicals. Increased peroxynitrite formation has been related to the pathogenesis of multiple diseases, thus underlining the importance of understanding the mechanisms of its detoxification. In nature, different enzymatic routes for peroxynitrite decomposition have evolved. Among them, peroxiredoxins catalytically reduce peroxynitrite in vitro; modulation of their expression affects peroxynitrite-mediated cytotoxicity, and their content changes in pathologic conditions associated with increased peroxynitrite formation in vivo, thus indicating a physiologic role of these enzymes in peroxynitrite reduction. Selenium-containing glutathione peroxidase also catalyzes peroxynitrite reduction, but its role in vivo is still a matter of debate. In selected cellular systems, heme proteins also play a role in peroxynitrite detoxification, such as its isomerization by oxyhemoglobin in red blood cells. Moreover, different pharmacologic approaches have been used to decrease the toxicity related to peroxynitrite formation. Manganese or iron porphyrins catalyze peroxynitrite decomposition, and their protective role in vivo has been confirmed in biologic systems. Glutathione peroxidase mimetics also rapidly reduce peroxynitrite, but their biologic role is less well established. Flavonoids, nitroxides, and tyrosine-containing peptides decreased peroxynitrite-mediated toxicity under different conditions, but their mechanism of action is indirect.

Ethanol induces peroxynitrite-mediated toxicity through inactivation of NADP +-dependent isocitrate dehydrogenase and superoxide dismutase

It has been reported that chronic alcohol administration increases peroxynitrite hepatotoxicity by enhancing concomitant production of nitric oxide and superoxide. Several studies have shown the importance of superoxide dismutase (SOD) in protecting cells against ethanol-induced oxidative stress. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of NADP +-dependent isocitrate dehydrogenase (ICDH) through to supply NADPH for antioxidant systems. In this report, we demonstrate that ethanol induces the peroxynitrite-mediated cytotoxicity in HepG2 cells through inactivation of antioxidant enzymes such as ICDH and SOD. Upon exposure to 100 mM ethanol for 3 days to HepG2 cells, a significant decrease in the viability and activities of ICDH and SOD was observed. The ethanol-induced inactivation of antioxidant enzymes resulted in the cellular oxidative damage and modulation of redox status as well as mitochondrial dysfunction in HepG2 cells. The cytoxicity of ethanol and inactivation of antioxidant enzymes were effectively protected by manganeses(III) tetrakis( N-methyl-2-pyridyl) porphyrin, a manganese SOD mimetic, and N′-monomethyl- l-arginine, a nitric oxide synthase inhibitor. These results indicate that ethanol toxicity is mediated by peroxynitrite and the peroxynitrite-mediated damage to ICDH and SOD may be resulted in the perturbation of the cellular antioxidant defense systems and subsequently lead to a pro-oxidant condition.

Role of endogenous glutathione in the secondary damage in experimental spinal cord injury in mice

GSH plays multiple roles in the nervous system including free radical scavenger, redox modulator of ionotropic receptor activity, and possible neurotransmitter. A lot of evidence suggests that GSH is involved in the pathogenesis of neurodegenerative disorders, like spinal cord injury (SCI). This study was undertaken to determine if the inhibition of endogenous glutathione, by L-buthionine-(S,R)-sulfoximine (BSO), affords protection against peroxynitrite-mediated toxicity in response to the spinal cord injury in vivo. The spinal cord of damaged animals showed a significant elevation of biochemical, immunohistochemical and functional parameters, increasing, respectively, neutrophils infiltration, lipid peroxidation, nitrotyrosine formation, PAR expression, apoptosis (measured by TUNEL staining) and loss of hind legs movement in SCI-operated mice. In contrast, the administration of BSO led to worsening of this already compromised setting, increasing the degree of (1) neutrophil infiltration, (2) lipid peroxidation, (3) histological damage, (4) apoptosis, (5) nitrotyrosine formation, (6) PAR expression, (7) apoptosis (measured by TUNEL staining) and (7) loss of hind legs movement. Thus, endogenous glutathione plays an important protective role against secondary damage after SCI.

Peroxynitrite and drug-dependent toxicity

Peroxynitrite is the product of the diffusion-controlled termination reaction between two radicals, nitric oxide and superoxide and is a strong oxidant and nitrating intermediate. Critical biomolecules like proteins, lipids and DNA react with peroxynitrite via direct or radical-mediated mechanisms, resulting in alterations in enzyme activities and signaling pathways. The biological consequences of peroxynitrite-mediated oxidative modifications depend on the levels of oxidant achieved in vivo and its cellular site of production. High and prolonged fluxes of peroxynitrite that overcome the endogenous antioxidant mechanisms, end up in disruption of cell homeostasis leading to apoptotic or necrotic cell death. Several drugs used in modern medicine and agriculture can exert their toxic side effects through mechanisms involving the formation of toxic levels of peroxynitrite, via redox cycling, uncoupling of nitric oxide synthase, stimulation of the endogenous formation of nitric oxide and superoxide or lowering of the antioxidant defenses. Experimental evidence point to peroxynitrite participation in the toxicity of doxorubicin, paraquat, acetaminophen and MPTP ( N-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine). The pharmacology against peroxynitrite-mediated toxicity could be oriented towards decreasing the levels of the precursor radicals (i.e. using NOS or oxidases inhibitors, SOD mimetics) or reducing the levels of peroxynitrite itself (peroxynitrite scavengers or decomposition catalysts) and serve to attenuate or neutralize drug-dependent toxicity linked to enhanced peroxynitrite formation.

Induction of endogenous glutathione by the chemoprotective agent, 3H-1,2-dithiole-3-thione, in human neuroblastoma SH-SY5Y cells affords protection against peroxynitrite-induced cytotoxicity

Substantial evidence suggests that peroxynitrite generated from the bi-radical reaction of nitric oxide and superoxide is critically involved in the pathogenesis of neurodegenerative disorders, such as Parkinson’s disease. Reaction with sulfhydryl (SH)-containing molecules has been proposed to be a major detoxification pathway of peroxynitrite in biological systems. This study was undertaken to determine if chemically elevated intracellular reduced glutathione (GSH), a major SH-containing biomolecule, affords protection against peroxynitrite-mediated toxicity in cultured neuronal cells. Incubation of human neuroblastoma SH-SY5Y cells with the unique chemoprotectant, 3H-1,2-dithiole-3-thione (D3T), led to a significant elevation of cellular GSH in a concentration-dependent fashion. To examine the protective effects of D3T-induced GSH on peroxynitrite-mediated toxicity, SH-SY5Y cells were pretreated with D3T and then exposed to either the peroxynitrite generator, 3-morpholinosydnonimine (SIN-1), or the authentic peroxynitrite. We observed that D3T-pretreated cells showed a markedly increased resistance to SIN-1- or authentic peroxynitrite-induced cytotoxicity, as assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium reduction assay. Conversely, depletion of cellular GSH by buthionine sulfoximine (BSO) caused a marked potentiation of SIN-1- or authentic peroxynitrite-mediated cytotoxicity. To further demonstrate the causal role for GSH induction in D3T-mediated cytoprotection, SH-SY5Y cells were co-treated with BSO to abolish D3T-induced GSH elevation. Co-treatment of the cells with BSO was found to significantly reverse the protective effects of D3T on SIN-1- or authentic peroxynitrite-elicited cytotoxicity. Taken together, this study demonstrates for the first time that D3T can induce GSH in cultured SH-SY5Y cells, and that the D3T-augmented cellular GSH defense affords a marked protection against peroxynitrite-induced toxicity in cultured human neuronal cells.

Ethanol Consumption Increases Nitric Oxide Production in Rats, and Its Peroxynitrite-Mediated Toxicity Is Attenuated by Polyenylphosphatidylcholine

Background: Nitric oxide generally mediates beneficial responses but becomes deleterious when coexistence with enhanced superoxide formation leads to the synthesis of peroxynitrite, a potent oxidant and nitrating agent. Methods: To study the effects of ethanol and polyenylphosphatidylcholine on nitric oxide metabolism and toxicity, 36 rats were pair-fed liquid diets with 36% of energy either as ethanol or as additional carbohydrate for 24 days and were killed 90 min after intragastric feeding. Half received polyenylphosphatidylcholine in the diet (3 g/liter), and the other half equivalent amounts of essential fatty acids and choline. Nitric oxide was measured by chemiluminescence in arterial blood and liver cytosol and as a product of the inducible nitric oxide synthase activity. Peroxynitrite formation was assessed by the increase in nitrotyrosine protein residues, measured immunochemically. Results: In blood, administration of ethanol with or without polyenylphosphatidylcholine doubled nitric oxide levels. In the liver, ethanol increased nitric oxide by 52% (p < 0.01), and polyenylphosphatidylcholine attenuated this effect. Ethanol consumption increased the cytosolic activity of the inducible nitric oxide synthase and induced microsomal cytochromes P-450 capable of producing both nitric oxide and superoxide. This was associated with an 18% (p < 0.01) increase in nitrotyrosine protein residues, products of peroxynitrite toxicity, which occurred predominantly in steatotic hepatocytes. Polyenylphosphatidylcholine attenuated these changes by decreasing the ethanol effect on both the cytosolic and the microsomal activities, in addition to acting as a powerful antioxidant. Acute administration of the same ethanol dose increased nitric oxide levels, but did not affect nitrotyrosine protein residues. Conclusions: Chronic, but not acute, ethanol administration increases peroxynitrite hepatotoxicity by enhancing concomitant production of nitric oxide and superoxide, both of which are prevented by polyenylphosphatidylcholine.

Ethanol Consumption Increases Nitric Oxide Production in Rats, and Its Peroxynitrite‐Mediated Toxicity Is Attenuated by Polyenylphosphatidylcholine

Nitric oxide generally mediates beneficial responses but becomes deleterious when coexistence with enhanced superoxide formation leads to the synthesis of peroxynitrite, a potent oxidant and nitrating agent. To study the effects of ethanol and polyenylphosphatidylcholine on nitric oxide metabolism and toxicity, 36 rats were pair-fed liquid diets with 36% of energy either as ethanol or as additional carbohydrate for 24 days and were killed 90 min after intragastric feeding. Half received polyenylphosphatidylcholine in the diet (3 g/liter), and the other half equivalent amounts of essential fatty acids and choline. Nitric oxide was measured by chemiluminescence in arterial blood and liver cytosol and as a product of the inducible nitric oxide synthase activity. Peroxynitrite formation was assessed by the increase in nitrotyrosine protein residues, measured immunochemically. In blood, administration of ethanol with or without polyenylphosphatidylcholine doubled nitric oxide levels. In the liver, ethanol increased nitric oxide by 52% (p < 0.01), and polyenylphosphatidylcholine attenuated this effect. Ethanol consumption increased the cytosolic activity of the inducible nitric oxide synthase and induced microsomal cytochromes P-450 capable of producing both nitric oxide and superoxide. This was associated with an 18% (p < 0.01) increase in nitrotyrosine protein residues, products of peroxynitrite toxicity, which occurred predominantly in steatotic hepatocytes. Polyenylphosphatidylcholine attenuated these changes by decreasing the ethanol effect on both the cytosolic and the microsomal activities, in addition to acting as a powerful antioxidant. Acute administration of the same ethanol dose increased nitric oxide levels, but did not affect nitrotyrosine protein residues. Chronic, but not acute, ethanol administration increases peroxynitrite hepatotoxicity by enhancing concomitant production of nitric oxide and superoxide, both of which are prevented by polyenylphosphatidylcholine.

Alcohol increases hepatic nitric oxide production and its peroxynitrite-mediated toxicity, which are prevented by polyenylphosphatidylcholine

Alcohol increases hepatic nitric oxide production and its peroxynitrite-mediated toxicity, which are prevented by polyenylphosphatidylcholine